1. Field of the Invention
The present invention relates generally to proteins which regulate genes and to methods for altering gene expression, cellular function and metabolism. In particular, the invention concerns nuclear proteins which bind sterol regulatory elements (SREs), such as the SRE-1 (SEQ ID NO:27, for example) of the low density lipoprotein (LDL) receptor gene, and methods for identifying candidate substances which stimulate SRE-1-mediated gene transcription. Such substances are contemplated for use in reducing plasma cholesterol levels and, thereby, for controlling hypercholesterolemia and its associated diseases.
2. Description of the Related Art
There is currently relatively little knowledge concerning feedback suppression mechanisms involved in eukaryotic gene regulation. In animal cells, most attention has focused on positively-regulated systems in which hormones, metabolic inducers, and developmental factors increase transcription of genes. These inducing agents are generally thought to activate or form complexes with proteins that stimulate transcription by binding to short sequences of 10 to 20 base pairs (bp) in the 5'-flanking region of the target gene. Such elements, termed GRE, MRE, and IRE have been reported for glucocorticoid, metal and interferon regulatory elements, respectively (Yamamoto, 1985; Stuart et al., 1984; Goodbourn et al., 1986).
Important advances have been made recently concerning other DNA segments which are capable of conferring control capability to known genes in eukaryotic systems. For example, transcription of the gene for the low density lipoprotein (LDL) receptor is regulated by a 10 base pair (bp) element in the 5' flanking region designated sterol regulatory element-1 (SRE-1, SEQ ID NO:27) (Goldstein and Brown, 1990; U.S. Pat. No. 4,935,363). The receptor provides cholesterol to cells by binding and internalizing LDL, a plasma cholesterol transport protein. When cellular cholesterol demands are high, as when cells are grown in the absence of sterols, this element is transcriptionally active, the cells produce large numbers of LDL receptors and LDL is internalized rapidly. On the other hand, when sterols accumulate within cells, the SRE-1 is silenced, and cells reduce the number of LDL receptors, thereby preventing cholesterol over accumulation. This feedback regulatory system controls not only the cholesterol content of cells, but also that of plasma (Brown and Goldstein, 1986). When hepatic LDL receptors are repressed by intracellular accumulation of dietary cholesterol, LDL is not taken up into the liver at a normal rate, and the lipoprotein builds up to high levels in the blood.
The 10 bp SRE-1 (SEQ ID NO:27) lies within a 16-base pair (bp) sequence, designated Repeat 2 (SEQ ID NO:22), that is 53 bp upstream of the transcription start site of the LDL receptor gene (Smith et al., 1990). This sequence is the central member of a series of three imperfect repeats in the 5' flanking region, (Repeats 1; 2, SEQ ID NO:22; and 3, SEQ ID NO:23), all of which are required for high level transcription (Goldstein and Brown, 1990; Smith et al., 1990; Sudhof et al., 1987). Repeats 1 and 3 (SEQ ID NO:23) bind Sp1, a constitutive transcription factor. Mutations in any of the three repeat sequences abolish high-level transcription in sterol-deprived cells (Smith et al., 1990; Sudhof et al., 1987; Dawson et al., 1988).
The activity of Repeats 1 and 3 (SEQ ID NO:23), although necessary, is not sufficient for high level transcription. An additional positive contribution is required from Repeat 2 (SEQ ID NO:22), which does not bind Sp1 (Smith et al., 1990; Sudhof et al., 1987; Dawson et al., 1988). Mutational analysis suggests that Repeat 2 (SEQ ID NO:22) binds a conditionally positive transcription factor that is active only under conditions of sterol deprivation (Smith et al., 1990). When sterols are added to cells, the contribution of Repeat 2 (SEQ ID NO:22) is abolished, and the rate of transcription falls.
The nucleotides within Repeat 2 (SEQ ID NO:22) that are necessary for its transcriptional activity have been delineated partially through in vitro mutagenesis and expression studies in permanently transfected CHO cells. The relevant nucleotides include the SRE-1 10 bp stretch which has the sequence ATCACCCCAC (SEQ ID NO:27) (Smith et al., 1990). The essential elements of this sequence have been shown to be conserved in evolution as far back as the last common ancestor of humans and frogs (Mehta et al., 1991).
Unfortunately, despite the elucidation of the SRE-1 DNA sequence (SEQ ID NO:27), the nature of the putative transcription factor that binds to SRE-1 remained unknown. Two candidates have been proposed (Rajavashisth et al., 1989; Stark et al., 1992), but the proteins in these reports did not show specific binding which precisely correlated with the transcriptional activity of modified SRE-1 elements, and purification of the putative binding proteins was not reported.
The identification of a protein which binds to the SRE-1 sequence and functions to promote transcription would be particularly advantageous. Not only would such a transcription factor be useful in terms of furthering an understanding of eukaryotic gene control in general, but it would also provide a powerful tool for directly and indirectly regulating specific gene expression. A purified SRE-1 binding protein would be extremely useful as the central component in screening assays to identify pharmacological agents capable of altering gene transcription, and particularly, substances capable of promoting LDL receptor gene transcription, especially those which do so even in the presence of sterols which normally down regulate the receptor. Such compounds would act as agents to reduce plasma LDL-cholesterol levels and would represent a significant medical breakthrough.